This invention relates to an improved method for measuring the freezing point lowering and also to a novel device being based on same, whereby and wherein a portion of a supercooled sample liquid is made to be changed further into a extra-supercooled state as a freezing stimulant to the whole supercooled sample liquid for the purpose of measuring the osmotic pressure of sample liquids, for example in particular, body fluids such as plasma, spinal fluid, tear and the like or dialyzed fluid as well as urine, which are the objects of clinical examination.
The living body is occupied by water up to about 60%. This water is present within the human body as plasma, cell fluid or the other body fluids in the state of dissolved substances of different species. Since the cell membrane of the living body is a semipermeable one, the osmotic pressure of the body fluids being dialyzed at the membrane has a great influence on the functions of the living body.
In consideration of such things, there have been recently brought to market one after another many kinds of devices for measuring the osmotic pressure of various body fluids or urine. The measurement of the osmotic pressure of plasma, urine, spinal fluid, etc. has come to be taken in daily examination, serving the purpose of diagnosing and controlling the patients who are suffering from dehydration, diabetes insipidus, hypochloremia, hyperchloremia, uremia, non-ketonic and high osmotic coma etc., etc. inclusive of the postoperative patients.
Seeking effective utilization, methods and devices for measuring the osmotic pressure should be able to operate on a dose of sample liquid being used for measuring the priceless spinal fluid which has been reduced or tear fluid to a possible infinitesimal. Such devices should also have the ability of quickly examining a great number of subject in a short time in an urgent need, and the possibility of the continuous measurement as of the dialyzed liquid which is inevitable at the time of the artificial dialysis.
However, the above-mentioned requirements are difficult to be fulfilled by any of a number of so-called devices for measuring the osmotic pressure now available on the market. Above all, it is impossible with them to perform the last referred-to continuous measurement. In addition, they contain further various points in question, as stated later. Under these circumstances, the appearance of a new type of method and device has been long eagerly waited for in this field.
The osmotic pressure in a solution is originally defined as the pressure produced by the diffusion of pure water into the solution which is partitioned by a semi-permeable membrane. In the case of a dilute solution, like its other properties such as the freezing point lowering, the boiling point elevation, and the vapor pressure lowering, the osmotic pressure is directly proportional to the number of moles of the solute contained in the solution itself. Directly, it is measured with the use of the semipermeable membrane, but indirectly, it is determined by converting the values derived from any of the above-stated other three properties.
In the case of the body fluid, however, protein in it brings about heat coagulation if osmotic pressure is measured by the boiling point elevation process, otherwise it gives rise to a considerable error if osmotic pressure is measured by the vapor pressure lowering process, particularly when some volatile constituent such as alcohol is contained therein. Also it is impossible for the direct method to find out a suitable semipermeable membrane, as a result of which it takes a lot of time in measuring. Accordingly, the freezing point lowering method has come to be exclusively adopted.
What is called here as the freezing point lowering means a phenomenon where the freezing point of a solution becomes lower than that of a pure solvent in proportion to the mole concentration of a solute contained in the solution, as mentioned above, which is widely utilized for the molecular weight determination. It is the case with the osmotic pressure. It also is proportional to the number of molecules (atoms) contained in the solution, so that it becomes possible to find the osmotic pressure by measuring the degree of freezing point lowering, as shown in the following formula: (I) ##EQU1## whereat .DELTA. Tf denotes the degree of freezing point lowering, R denotes the gas constant, Tf denotes the freezing point of the solvent (absolute temperature), Io denotes the melting latent heat per gram of the solvent, C denotes the molar number of the solute dissolved in 1 Kg of the solvent, and Kf denotes the depression of molar freezing point.
In the case where the solvent is water as the body fluid, the degree of freezing point lowering of the solution of the molar concentration 1 mol/Kg amounts to 1.858.degree. C. (Kf=1.858.degree. C.), and the freezing point is at -1.858.degree. C. Contrary to this, when the freezing point of the solution reads -1.857.degree. C., the molar concentration of it becomes 1 mol/Kg. Since the unit of osmotic pressure is the Osmol, the osmotic pressure of the solution of the molar concentration 1 mol/Kg can be expressed by 1 Osm/Kg. However, the osmotic pressure in the body fluid varies so very slightly that it is adequate to use the unit of 1:1000 of 1 Osm/Kg, that is, 1 m Osm.
Devices for measuring the osmotic pressure now in use all are composed of, for example, as disclosed in U.S. Pat. No. 3,203,226, a cooling tub to bring sample liquid into the supercooled state lower by several degrees than the freezing point, a head consisting of a bar thermistor for use in detecting the temperature of the freezing point and an electromagnetic vibratory stirring rod for the freezing, a measuring circuit, a controlling part, an indicating part, and others. In such a composition, the thermistor and the stirring rod are inserted into a test tube (discrete cell) containing the sample liquid. This test tube is dipped into the cooling tub to bring the sample liquid into a supercooled state. When the temperature of the sample liquid falls to -5.degree..about.-6.degree. C., the stirring rod is forced to hastily vibrate to generate cryohydrate nuclei therein, through which the sample liquid as a whole is caused to be frozen. At this time, the temperature of the sample liquid rises to the temperature of the freezing point through the evolution of the freezing latent heat, and is kept in the state of flatness for some time. After a while, it begins to fall gradually. The temperature at this flat part (plateau), that is, the temperature of the sample liquid which is in the state of coexistence of solid and liquid phases, is detected by means of the thermistor and displayed in the form of an osmotic pressure after being converted on the basis of the degree of freezing point lowering.
As stated above, existing apparatuses and methods of this kind can make the freezing point distinct by congealing the sample liquid after being supercooled while giving it a vibrative freezing stimulation, and they can measure the osmotic pressure with accuracy and in a relative short time with the use of a thermistor which is of a little heat capacity and able to detect exactly a slight difference in termperature. Inasmuch as a stirring rod is used for the freezing stimulation, however, they have still the following disadvantages:
(1) It is necessary to insert at the same time both the stirring rod and the thermistor for measuring the freezing point into the sample liquid, and consequently a relatively large-sized test tube must be used for it, as a result of which it is hard to reduce the sample tested to a microquantity. Even if the stirring rod and the thermistor both are tried to be miniaturized, a space of a certain extent is necessary all the same. Furthermore, there is a limit in such a miniaturization for the following reason:
In intensity of freezing vibration of the stirring rod is so considerably strong that the outside air is apt to be sucked into the sample liquid on occasion. Although the vibration time is as short as only about 1 second, the vibration in the progress of freezing exerts pressure on the thermistor amidst the state of coexistence of solid and liquid phases of the sample. As a result, restrictions are placed on the miniaturization of both the thermistor and the stirring rod from the viewpoint of the strength.
(2) The difficulty of miniaturizing the test tube and of reducing the sample tested to a microquantity is inevitably followed by having to make the compression refrigerating machine, Patier element, and others large-sized in order to enhance the cooling power, in company with which a large-sized means for air-cooling or water-cooling is also needed in order to exhaust efficiently the heat generated within the cooling means, therefore the prior apparatus as a whole is compelled to be made large-sized.
(3) In conventional apparatuses being equipped with a cooling tub using liquid refrigerant, the process and structure are apt to become complicated because there is a necessity now of gently stirring the sample liquid while cooling it with a view to making the measurement of the freeezing point easy by bringing the sample into a supercooled state as fast as possible. On the other hand it is desirable to elongate the plateau after freezing while keeping the temperature of the sample liquid constant. The sample is cooled uniformly by hastily cooling the cell-body until it reaches a suitable temperature while dipping it in liquid refrigerant and then slowly cooling the cell body while keeping it in cold air after pulling it up out of the liquid refrigerant.
(4) In these apparatuses, the stirring rod is exclusively employed as a freezing means, wherefore it is difficult for them to use a flow cell, and consequently to perform the continuous measurement or to measure a number of subjects in a short time. Existing apparatus all are in need of the respective tubes for each sample. In case of having constructed a kind of apparatus for the continuous measurement of one and the same sample, it is necessary for it to quantitatively pipet the sample liquid into the test tube each occasion, in consequence of which some specially-made pipetting means and transfer mechanism of test tubes come to be required, thereby entailing a flaw that the apparatus as a whole becomes structurally complicated and large-sized.